Brain transcription factors, nucleic acids encoding same and uses thereof

ABSTRACT

This invention provides an isolated, animal nucleic acid molecule encoding the Brain Factor-1, Brain Factor-2 and Brain Factor-3. This invention also provides expression vectors containing these nucleic acid molecules, host vector systems containing the vectors and a method of producing the Brain factor comprising growing the host vector system under suitable conditions. This invention also provides a DNA vector which comprises the 5&#39; nontranscribed region of the Brain Factor-1 gene, 3&#39; nontranscribed region of the Brain Factor-1 gene and a gene of interest, linked operably.

This application is a continuation-in-part of U.S. application Ser. No. 07/882,292, filed May 13, 1992, now U.S. Pat. No. 5,324,638 the contents of which are hereby incorporated by reference.

BACKGROUND OF THE INVENTION

Throughout this application various references are referred to within parenthesis. Disclosures of these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains. Full bibliographic citation for these references may be found at the end of this application, preceding the sequence listing and the claims.

One of the important early events in the development of the mammalian central nervous system is the establishment of regional diversity along the neural tube under the influence of signals from the underlying mesoderm (Mangold 1933; Spemann 1938; Placzek et al. 1990). The caudal portion of the neural tube gives rise to the spinal cord while the rostral part develops into the brain. Morphologic studies have identified the initial subdivision of the developing brain into three vesicles, the forebrain, midbrain and hindbrain. The forebrain and hindbrain subsequently each subdivide, resulting in the five major regions of the brain. The most rostral region, the telencephalon, gives rise to the structures of the cerebral hemispheres which include the cerebral cortex and basal ganglia (Kandel et al. 1991). This subdivision of the developing brain is apparent initially as changes in the shape of the neuroepithelium, and is established prior to differentiation of the progenitor cells into neurons and glia (McKay 1989).

Our understanding of the molecular events which establish the regional subdivision of the brain during mammalian development have been aided by several approaches. Because development depends on the expression of genes in distinct spatial and temporal patterns, one approach has been the study of transcription factors which control gene expression in a cell or tissue-specific fashion. Another approach is the identification of mammalian homologs of Drosophila genes which have been established to play crucial roles in insect development (Kessel and Gruss 1990). For example, homeobox gene complexes in Drosophila and mammals are strikingly similar in structural organization and expression pattern along the A-P axis of the organism (Duboule and Dolle 1989; Graham et al. 1989). While recent findings of restricted expression in the developing brain of several homeodomain and one zinc finger proteins have provided insight into the development of the hindbrain (Murphy et al. 1989; Wilkinson et al. 1989; Wilkinson et al. 1989; Hunt et al. 1991). The molecular basis of forebrain development remains poorly understood.

We have recently described a novel family of transcriptional activators, the HNF-3 family, whose members function to stimulate expression of a group of liver-specific genes. The expression of these factors in the mature animal is limited to tissues which derive from the gut endoderm such as the liver, lung and intestine (Lai et al. 1991). These findings suggested that HNF-3 family members might play a significant role in the development of these tissues. This hypothesis was supported when we learned that HNF-3 proteins were highly homologous to a Drosophila protein, fork head, which had been shown to be critical to normal fly development and to be expressed in the cells which were to form the insect gut structures (Weigel et al. 1989; Weigel and Jackle 1990). In this paper we described the discovery of a new member of the HNF-3/fork head family of transcription factors which is unique in that its expression is restricted to the telencephalon of the developing rat brain. Expression is readily detectable by embryonic day 10 in the area of the neural tube which gives rise to the telencephalic vesicles suggesting a critical role for this factor in the development of this region of the forebrain.

SUMMARY OF THE INVENTION

This invention provides an isolated, animal nucleic acid molecule encoding the Brain Factor-1, Brain Factor-2 and Brain Factor-3.

This invention also provides methods of detecting the expression of these Brain Factors. This invention also provides expression vectors to the Brain Factors.

This invention provides purified, animal Brain Factor-2 and animal Brain Factor-3.

This invention also provides antibodies of these Brain Factors and the uses of these antibodies to detect Brain Factors.

Finally, this invention provides a DNA vector which comprises the 5' nontranscribed region of the Brain Factor-1 gene, 3' nontranscribed region of the Brain Factor-1 gene and a gene of interest, linked operably.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C Nucleic acid sequence of BF-1 (Seq.I.D. Nos. 1 and 2). The protein sequence is shown below beginning with the initiator methionine at base 443. The DNA binding domain homology extends from amino acid 162-271. Underlined amino acids are identical to those in the DNA binding domain of at least two of the four previously identified members of the HNF-3/fork head family.

FIG. 2 Translation of the BF-1 protein in reticulocyte lysates from transcripts synthesized in vitro from the full length BF-1 template (lane 1), or templates deleting the 5' end of the cDNA 150 bp, BF-1Δ150 (lane 2) or 331 bp, BF-1Δ331 (lane 3). The size of the major product is the same for all three, demonstrating that the initiation site lies downstream of nucleotide 331. The lower MW products represent internal initiations, while the minor higher MW product present with templates BF-1 and BF-1Δ150 represents initiation at an upstream GUG at nucleotide 254. As expected, this higher MW product is absent with template BF-1Δ331. These additional products are attributable to the more permissive initiations which occur in reticulocyte lysates.

FIG. 3 Northern analysis of poly A+ RNA from adult (A) and E17 fetal (F) rat tissues. One major mRNA species is detected in the brain at 2.9 kB, with a probe for BF-1. A minor band is seen at 5 kB. This mRNA is not detected in the other tissues even with prolonged exposure of the blot. The same blot was reprobed with GAPDH to assess the quantity of RNA. Sm INT--small intestine.

FIGS. 4A and B Ribonuclease protection assays of total RNA from rat tissues. A 309 nucleotide fragment is specifically protected by the BF-1 mRNA. A) The protected band is detectable only in the brain. M--molecular weight markers; I--small intestine; L--liver; B--brain; S--spleen, K--kidney; T--testis. B) RNA prepared from different regions of the adult brain, adrenal gland, and whole brain or liver from four different stages of development. CAUD--caudate putamen; Fr CORT--frontal cortex; Oc CORT--occipital cortex; CEREB--cerebellum; OLF--olfactory bulb; HIPP--hippocampus; THAL--thalamus; Ad--adult; NB--newborn; E17-embryonic day 17; E14-embryonic day 14; BR-brain; LIV-liver.

FIGS. 5A-5K Expression pattern of BF-1 during rat development assessed by in-situ hybridization with an antisense probe (A-H, J-M). Pairs of photomicrographs and the corresponding autoradiograph from a transverse section of an E11.5 embryo (A, B), sagittal section of an E15 embryo (C, D) and a sagittal section of an E17 brian (E, F). Arrowheads in E indicate the approximate level of coronal sections (G,H) whose autoradiographs are shown. I is a representative control autoradiograph of a section parallel to that shown in E, hybridized with the corresponding sense probe for BF-1. J and K are autoradiographs of sagittal and coronal sections from adult brain. t, Telencephalon; d, diencephalon; ms, mesencephalon; mt, metencephalon; my, myelencephalon; s, spinal cord; cor, cerebral cortex; cb, cerebellum; cp, caudate putamen; h, hippocampus; tv, telencephalic vesicle; lv, lateral ventricles; op, optic stalk; r, retinal neuroepithelium; olf, olfactory placode; ole, olfactory epithelium; fg, foregut.

FIGS. 6A-6C Restriction of BF-1 expression to the telecephalon of the developing brain. Brightfield and darkfield photomicrographs of sections hybridized with a BF-1 antisense probe. A an B) Sagittal section from E10 with hybridization signal over the rostral end of the neural tube. C and B) Parasagittal section from E10 again with signal over the rostral neural tube as well as the auditory vesicle and pharyngeal pouches. C) Transverse section from E11.5 caudal to the section shown in FIG. 5A. t, telencephalon; d, diencephalon; Ms, mesencephalon; av, auditory vesicle; php, pharyngeal pouches; olf, olfactory placode; r, retinal neuroepithelium; sb, superior bridge.

FIGS. 7A and 7D Restriction of BF-1 expression to the telencephalon at embryonic day 13.5. Brightfield and darkfield photomicrographs of sections hybridized with a BF-1 antisense probe. A and B) Transverse section from E13.5 with hybridization signal over the cerebral cortical neuroepithelium, the basal telencephalic neuroepithelium and the differentiating basal telencephalon. C and D) Transverse section rostral to that in A with hybridization over the cerebral cortical neuroepithelium. No detectable signal over the thalamic and hypothalamic neuroepithelium. cc, Cerebral cortical neuroepithelium; bt, basal telencephalic neuroepithelium; BT, basal telencephalon(Differentiating); inf, infundibulum; th, thalamus; hy, hypothalamus; my, myelencephalon; lv, lateral ventricle; v3, third ventricle.

FIG. 8 Sequence specific binding of BF-1. Autoradiograph of a gel mobility shift assay comparing two BF-1 fusion proteins and liver extract using two different labeled probes, B2 site from the HNF-1 promoter and TTR from the transthyretin promoter. 1) BF-1 pGEX-DNA complex with the B2 probe, 2) BF-1 pET-DNA complex with the B2 probe, 3) the three complexes in liver extract from HNF-3a, 3b and 3g with both B2 and TTR probes. Alternate lanes include (-) no competitor or (+) 40-fold molar excess of unlabeled homologous competitor.

FIG. 9 Restriction map of BF-1 genomic DNA and two alternatively spliced cDNA.

FIGS. 10A and 10B BF-2 (Brain Factor-2) cDNA sequence (Seq.I.D. No.3).

FIG. 11 BF-3 (Brain Factor-3) cDNA sequence (Seq.I.D. No.4).

FIG. 12 Rat BF-1 cDNA Maps. This cDNA was cloned into EcoRI and NotI sites of pBluescript KS plasmid vector and can be excised using these restriction enzymes. The plasmid made is designated, pBSKS-BF-1. Also shown are additional unique restriction sites. The map is not drawn to scale.

FIG. 13 Rat BF-2 cDNA Maps. This cDNA was cloned into EcoRI and NotI sites of pBluescript KS plasmid vector and can be excised using these restriction enzymes. The plasmid made is designated, pBSKS-BF-2. Also shown are additional unique restriction sites. The map is not drawn to scale.

FIG. 14 Rat BF-3 cDNA Maps. This cDNA were cloned into EcoRI and NotI sites of pBluescript KS plasmid vector and can be excised using these restriction enzymes. The plasmid made is designated, BF-3. Also shown are additional unique restriction sites. The map is not drawn to scale.

FIG. 15 Map of the Bf-1-lacZ expression cassette in plasmid pTBL3. Sfil and Xhol linkers were introduced into the Sphl and EcoRI sites of the vector, pGEM4 respectively. Then whole BF-1-lacZ expression cassette was cloned into the Sfil and Xhol sites of the modified vector. Ncol and Kpnl are not unique sites.

FIG. 16 Western analysis of BF-1 peptide antiserum. Partially purified BF-1 fusion protein run on SDS-PAGE gel and transferred to nitrocellulose. Lanes 1, 3, 5, 7 have 100 ng and lanes 2, 4, 6, 8 have 5 ng of BF-1. Lanes 1-2)1:2000 pre-immune serum. Lanes 3-4)1:20,000 pre-immune serum. Lanes 5-6)1:2000 BF-1 peptide antiserum. Lanes 7-8)1:20,000 BF-1 peptide anti serum. Arrowhead marks position of BF-1 fusion protein. The lower band represents a proteolytic fragment.

FIG. 17 SDS-PAGE analysis of immunoprecipitates of S-35 labeled reticulocyte lysates containing BF-1 incubated with rabbit serum; lane 1 preimmune serum; lane 2 BF-1 peptide antiserum. BF-1 bound to antibodies are precipitated with Staph A-agarose beads.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides an isolated, animal nucleic acid molecule encoding the Brain Factor-1. This invention also provides an isolated, animal nucleic acid molecule encoding the Brain Factor-2. This invention further provides an isolated, animal nucleic acid molecule encoding the Brain Factor-3.

In an embodiment, the above described nucleic acid molecules are RNA. In another embodiment, the nucleic acid molecules are DNA. In a further embodiment, the DNA molecules are genomic. In a still further embodiment, the DNA molecules are cDNAs.

As used herein, the Brain Factors are Brain Factor-1, Brain Factor-2 and Brain Factor-3.

In an embodiment, the cDNA molecule has the nucleotide sequence substantially the same as the nucleotide sequence shown in FIGS. 1A-1C (Seq. I.D. No. 1). In another embodiment, the cDNA has the nucleotide sequence substantially the same as the nucleotide sequence shown in FIGS. 10A and 10B (Seq.I.D. No.3). In a further embodiment, the cDNA has the nucleotide sequence substantially the same as the nucleotide sequence shown in FIG. 11 (Seq.I.D. No.4).

This invention also provides plasmids which comprise the above described cDNA molecules.

In an embodiment, the rat BF-1 cDNA is cloned into the EcoRI and NotI sites of pBluescript KS plasmid and the resulting plasmid is designated, pBSKS-BF-1. This plasmid, pBSKS-BF-1, was deposited on Apr. 30, 1993 with the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Md. 20852, U.S.A. under the provisions of the Budapest Treaty for the International Recognition of the Deposit of Microorganism for the Purposes of Patent Procedure. Plasmid, pBSKS-BF-1, was accorded ATCC Accession Number 75459.

In another embodiment, the rat BF-2 is cloned into the EcoRI and NotI sites of pBluescript KS plasmid and the resulting plasmid is designated, pBSKS-BF-2. This plasmid, pBSKS-BF-2, was deposited on Apr. 30, 1993 with the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Md. 20852, U.S.A. under the provisions of the Budapest Treaty for the International Recognition of the Deposit of Microorganism for the Purposes of Patent Procedure. Plasmid, pBSKS-BF-2, was accorded ATCC Accession Number 75460.

In still another embodiment, the rat BF-3 cDNA is cloned into the EcoRI and NotI sites of pBluescript KS plasmid and the resulting plasmid is designated, BF-3. This plasmid, BF-3, was deposited on Apr. 30, 1993 with the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Md. 20852, U.S.A. under the provisions of the Budapest Treaty for the International Recognition of the Deposit of Microorganism for the Purposes of Patent Procedure. Plasmid, BF-3, was accorded ATCC Accession Number 75461.

This invention also provides a nucleic acid probe comprising a nucleic acid molecule of about 15 nucleotides capable of specifically hybridizing with a sequence included within the sequence of a nucleic acid molecule encoding Brain Factor-1, Brain Factor-2 or Brain Factor-3.

The nucleic acid probe produced can either be DNA or RNA. As used herein, the phrase "specifically hybridizing" means the ability of a nucleic acid molecule to recognize a nucleic acid sequence complementary to its own and to form double-helical segments through hydrogen bonding between complementary base pairs. Nucleic acid probe technology is well known to those skilled in the art who will readily appreciate that such probes may vary greatly in length and may be labeled with a detectable label, such as a radioisotope or fluorescent dye, to facilitate detection of the probe. DNA probe molecules may be produced by insertion of a DNA molecule which encodes the Brain Factor into suitable vectors, such as plasmids or bacteriophages, followed by transforming into suitable bacterial host cells, replication in the transformed bacterial host cells and harvesting of the DNA probes, using methods well known in the art. Alternatively, probes may be generated chemically from DNA synthesizers.

RNA probes may be generated by inserting a Brain Factor sequence downstream of a bacteriophage promoter such as T3, T7 or SP6. Large amounts of RNA probe may be produced by incubating the labeled nucleotides with the linearized Brain Factor fragment where it contains an upstream promoter in the presence of the appropriate RNA polymerase.

The current invention further provides a method to measure the expression of a Brain Factor in a cell. The probes synthesized above may be used to detect expression of a Brain Factor by detecting the presence of mRNA coding for the Brain Factor. Total mRNA from the cell may be isolated and contacting the mRNA so obtained with Brain Factor nucleic acid probe under hybridizing conditions. The presence of mRNA hybridized to the probe can be determined by gel electrophoresis or other methods known in the art. By measuring the amount of the hybrid made, the expression of the Brain Factor by the cell can be determined.

In one embodiment of this invention, nucleic acids are extracted by precipitation from lysed cells and the mRNA is isolated from the extract using an oligo-dT column which binds the poly-A tails of the mRNA molecules. The mRNA is then exposed to radioactively labelled probe on a nitrocellulose membrane, and the probe hybridizes to and thereby labels complementary mRNA sequences. Binding may be detected by luminescence autoradiography or scintillation counting. However, other methods for performing these steps are well known to those skilled in the art, and the discussion above is merely an example.

This invention further provides another method to measure the expression of the Brain Factors. The probes are also useful for in-situ hybridization or in order to locate tissues which express this gene, or for other hybridization assays for the presence of this gene or its mRNA in various biological tissues. The in-situ hybridization using a nucleic acid probe is well known in the art. Essentially, tissue sections are incubated with the nucleic acid probe to allow the hybridization to occur. Since the probe will carry a marker for the detection, the amount of the hybrid will be determined and so will the expression of the Brain Factor.

This invention further provides a method of detecting the expression of a Brain Factor in an animal tissue which comprises: (i) obtaining a sample from the animal tissue; (ii) contacting the sample with a labelled probe of either Brain Factor-1, Brain Factor-2 or Brain Factor-3 under conditions permitting the probe to bind to mRNA encoding the Brain Factor; (iii) removing unbound probe from the sample; and (iv) detecting the presence of bound, labelled probe in the sample, said presence indicating the expression of the Brain Factor in the animal tissue.

This invention also provide a method of detecting expression of a Brain Factor in an animal tissue by detecting the presence of mRNA coding for the Brain Factor which comprises: (i) obtaining mRNA sample from the animal tissue; (ii) contacting the obtained mRNA sample with a labelled probe of Brain Factor-b 1, Brain Factor-2 or Brain Factor-3 under conditions permitting the probe to bind to mRNA encoding the Brain Factor; (iii) removing unbound probe from the sample; and (iv) detecting the presence of bound, labelled probe in the sample, said presence indicating the expression of the Brain Factor in the animal tissue.

This invention also provides a method of diagnosing the abnormal expression of Brain Factor-1 in telencephalon-derived tissue or a tumor tissue from an animal which comprises the steps of:(i) obtaining a sample of telencephalon-derived tissue or a sample of tumor from the animal; (ii) contacting the tissue sample with a labelled probe of Brain Factor-1 conditions permitting the probe to bind to mRNA in the sample; (iii) removing unbound probe from the sample; (iv) detecting the presence of bound, labelled probe in the sample; (v) quantifying the amount of bound, labelled probe in the sample; and (vi) comparing the amount of bound, labelled probe in the sample to the amount of labelled probe which binds to a sample of non telencephalon-derived tissue or of telencephalon-derived tissue from a subject which expresses normal levels of the Brain Factor-1 in such tissue.

This invention also provides a method of diagnosing the abnormal expression of Brain Factor-1 in telencephalon-derived tissue or a tumor tissue from an animal which comprises:(i)obtaining mRNA sample from the telencephalon-derived tissue or tumor tissue; (ii) contacting the obtained mRNA sample with a labelled probe of Brain Factor-1, under conditions permitting the probe to bind to mRNA encoding the Brain Factor;(iii) removing unbound probe from the sample;(iv) detecting the presence of bound, labelled-probe in the sample;(v) quantifying the amount of bound, labelled probe in the sample; and(vi) comparing the amount of bound, labelled probe in the sample to the amount of labelled probe which binds to a sample of non telencephalon-derived tissue or of telencephalon-derived tissue from an animal which expresses normal levels of the Brain Factor-1 in such tissue.

This invention provides an expression vector comprising the genomic DNA molecule of Brain Factor-1, Brain Factor-2 or Brain Factor-3 operably linked to a promoter.

This invention provides an expression vector comprising the cDNA molecule of Brain Factor-1, Brain Factor-2 or Brain Factor-3 operably linked to a promoter.

This invention also provides a host vector system comprising the above-described expression vectors in a suitable host cell. In an embodiment, the suitable host is a bacterial cell. In another embodiment, the suitable host is a eukaryotic cell.

This invention also provides a method of producing a Brain Factor which comprises growing the above-described host vector systems under conditions which permit transcription and translation, followed by recovering the protein so produced.

This invention also provides a purified, animal Brain Factor-1, a purified, animal Brain Factor-2 and a purified, animal Brain Factor-3. In an embodiment, the purified, animal Brain Factor is mammalian. In a further embodiment, the purified, mammlian Brain Factor is from mouse, rat or human.

This invention further provides a protein which comprises an amino acid sequence fused to the purified the above-described purified Brain Factor or to a fragment of the purified Brain Factor.

The above fusion protein may be generated by chemical or genetical engineering methods. For example, the DNA sequence encoding a Brain factor or a fragment of the Brain Factor can be ligated to another DNA molecules which encoding another protein of interest. Therefore, the ligated molecule will produce a fusion protein which have some or all of the Brain Factor protein.

This invention also provides a pharmaceutical composition comprising the above-described purified Brain Factor and a pharmaceutically acceptable carrier.

This invention provides a method of correcting an animal's defective synthesis of a Brain Factor which comprises administering to the patient an effective amount of the above pharmaceutical composition.

This invention provides a method of correcting an animal's defective synthesis of a Brain Factor which comprises: (i) isolating suitable cells from the animal; (ii) inserting the expression vector comprising the genomic or complementary DNA molecule of Brain Factor-1, Brain Factor-2 or Brain Factor-3 operably linked to a promoter into the cells under conditions permitting stable integration of the vector into the genome of the cells and expression of the DNA molecule; and (iii) reintroducing the cells into the animal from which they were isolated.

This invention also provides a method of correcting an animal's defective synthesis of a Brain Factor which comprises: (i) introducing the expression vector comprising the genomic or complementary DNA molecule of Brain Factor-1, Brain Factor-2 or Brain Factor-3 operably linked to a promoter into a neuroepithelial cell line under conditions permitting stable integration of the vector into the genome of the neuroepithelial cells; and (ii) administering the neuroepithelial cells to the animal under conditions permitting the cells to grow in the animal and synthesize the Brain Factor encoded by the expression vector.

This invention also provide an antibody which specifically recognizes the Brain Factor-1, Brain Factor-2 or Brain Factor-3. In an embodiment, the above antibody is monoclonal. This invention also provides a hybridoma cell which produces the monoclonal antibody which specifically recognizes the Brain Factor-1, Brain Factor-2 or Brain Factor-3.

As an example to produce polyclonal antibodies to BF-1, a 15 aa peptide corresponding to amino acids 161-174 plus a cysteine residue was synthesized, purified by HPLC and coupled to the carrier, keyhole limpet hemocyanin (KLH) and injected into rabbits with adjuvant at 4-week intervals.

This peptide was chosen for its hydrophilicity and because it is not conserved between any of the known members of the gene family. Rabbits were sacrificed after four injections. Initial studies show this antisera reacts with BF-1 as tested by Western blot, using bactrially expressed BF-1 protein and immunoprecipitation of BF-1 protein translated in reticulocyte lysates (FIG. 16). This antisera can also immunoprecipitate BF-1 (FIG. 17).

This invention further provides a method of producing a monoclonal antibody which specifically recognizes a Brain Factor which comprises culturing the above-described hybridoma cell under conditions permitting antibody production, followed by recovering the antibody so produced. This invention also provide the above antibody labelled with a detectable marker.

This invention further provides a method of detecting the expression of a Brain Factor in an animal tissue which comprises:(i) isolating a sample of the animal tissue; (ii) contacting the sample with the above-described labelled antibody under conditions permitting the antibody to bind to the Brain Factor in the sample;(iii) removing unbound, labelled antibody from the sample; and (iv) detecting the presence of bound, labelled antibody in the sample, said presence indicating the presence of Brain Factor in the tissue.

This invention provides a method of detecting expression of a Brain Factor in a tumor comprises the steps of: (i) obtaining a sample of the tumor; (ii) contacting the sample with the above-described labelled antibody under conditions permitting the antibody to bind to the Brain Factor in the sample; (iii) removing unbound, labelled antibody from the sample; and (iv) detecting the presence of bound, labelled antibody in the sample, said presence indicating the presence of Brain Factor in the tumor.

This invention also provides a DNA vector which comprises the 5' nontranscribed region of the Brain Factor-1 gene, 3' nontranscribed region of the Brain Factor-1 gene and a gene of interest, linked operably.

This invention also provides a plasmid which comprises the above vector. In an embodiment, the plasmid is designated pTBL3. This plasmid, pTBL3, was deposited on Apr. 30, 1993 with the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Md. 20852, U.S.A. under the provisions of the Budapest Treaty for the International Recognition of the Deposit of Microorganism for the Purposes of Patent Procedure. Plasmid, pTBL3, was accorded ATCC Accession Number 75462.

This invention further provides a retroviral DNA vector which comprises the 5' nontranscribed region of the Brain Factor-1 gene, 3' nontranscribed region of the Brain Factor-1 gene and a gene of interest, linked operably.

This invention further provides the above-described retroviral vector, wherein the vector comprises DNA from a retrovirus corresponding to a 5' long terminal repeat, a 3' long terminal repeat, and a packaging signal.

This invention also provides plasmids which comprise the above-described retroviral vectors.

This invention provides an isolated, purified transcription factor expressed in the telencephalic region of the brain of a developing animal or in structures derived therefrom. For the purposes of this invention, an "isolated, purified" transcription factor is a nonnaturally occurring transcription factor, i.e., a transcription factor in a form which does not occur in nature. The term "transcription factor" as used herein means a protein whose presence is required to assist eucaryotic polymerases in starting RNA synthesis at correct sites on the DNA. The transcription factor may be a RNA polymerase II transcription factor, i.e., a factor which enables RNA polymerase II to start synthesis of mRNA at correct sites on the DNA. In one embodiment of this invention, the animal is a mammal, e.g., a mouse, rat or human.

The transcription factor of this invention is expressed in the telencephalon of the embryo of an animal and is also expressed, at a lower level, in the telencephalon-derived structures in the adult. The transcription factor of this invention is expressed in the cerebral hemispheres of the adult brian. The isolated, purified transcription factor provided by this invention may have the amino acid sequence shown in FIGS. 1 (Seq.I.D.Nos.1 and 2).

This invention also provides a protein which comprises an amino acid sequence fused to the isolated, purified transcription factor of claim 1 or to a fragment of the transcription factor. In one embodiment of this invention, the amino acid sequence is derived from glutathione transferase. In the presently preferred embodiment of this invention, the fragment of the transcription factor to which the amino acid sequence is fused corresponds to the DNA birding domain of the transcription factor.

This invention further provides a pharmaceutical composition comprising the transcription factor of this invention and a suitable carrier. Suitable carriers from a transcription factor are well known to those of ordinary skill in the art and include, but are not limited to, aqueous buffers. This invention still further provides a method of correcting an animal's defective synthesis of a transcription factor expressed in telencephalon-derived structures which comprises administering to the animal an effective amount of the pharmaceutical composition of this invention. "Correcting" an animal's defective synthesis of a transcription factor as used herein is intended to mean supplying the animal with an exogenous source of transcription factor when the animal itself synthesizes an insufficient amount of the transcription factor to meet its physiological needs. An "effective amount" of the pharmaceutical compositions is any amount of the composition effective to supply the animal with the transcription factor which the animal synthesizes in insufficient amounts. Methods of determining an effective amount are well known to those skilled in the art or can readily be determined by routine experimentation.

This invention provides an isolated nucleic acid molecule encoding the transcription factor of this invention. In one embodiment of this invention, the nucleic acid molecule is a messenger RNA molecule. In another embodiment of this invention, the nucleic acid molecule is a DNA molecule. The DNA molecule of this invention may be a cDNA molecule, e.g., a cDNA molecule having a nucleotide sequence substantially the same as the nucleotide sequence shown in FIG. 1 (Seq.I.D. No.1). The cDNA molecule may be labelled with a detectable marker, e.g., a radioisotope. The DNA molecule of this invention may also be a genomic DNA molecule.

Applicants have isolated a genomic DNA molecule which is approximately 20 kb in length and which includes all of the exons of the gene for the transcription factor. The genomic DNA molecule provided by this invention contains, in addition to the exons, DNA sequences which are involved with regulating the expression of the gene. These DNA sequences may be sequences to which factors bind that either promote or inhibit the expression of the gene. An example of such a factor is a transcription factor such as steroid hormone receptor. Accordingly, "correcting" an animal's defective synthesis of a transcription factor as used herein also means modulating the expression of the gene encoding the transcription factor. Such expression may be modulated by administering factors to the animal which either promote or inhibit expression of the gene. The ability of factors to promote or inhibit gene expression may be dependent on their ability to bind to DNA sequences which regulate gene expression. Such sequences involved in the regulation of the gene encoding the transcription factor of this invention are present in applicant's isolated genomic DNA molecule.

This invention provides a method of detecting the expression in the telencephalon-derived tissue of an animal of a transcription factor normally expressed in such tissue which comprises the steps of: isolating a sample of telencephalon-derived tissue from the animal; contacting the tissue sample with a labelled probe derived from the cDNA molecule of this invention under conditions permitting the probe to bind to mRNA encoding the transcription factor; removing unbound probe from the tissue sample; and detecting the presence of bound, labelled probe in the tissue sample, said presence indicating the expression of the transcription factor in the telencephalon-derived tissue. The animal may be a mammal, e.g., a mouse, rat or human. Methods of isolating telencephalon derive tissue from an animal are well known to those skilled in the art. Methods of contacting tissue samples with cDNA probes, conditions suitable for binding of cDNA probes to mRNA in a tissue sample and methods of removing unbound probe from a sample are also well known to those skilled in the art. Additionally, methods of detecting bound, labelled probe in a tissue sample are well known to those skilled in the art.

This invention also provides a method of diagnosing the abnormal expression of a transcription factor normally expressed in telencephalon-derived tissue which comprises the steps of: isolating a sample of tissue from the animal; contacting the tissue sample with a labelled probe derived from the cDNA molecule of this invention under conditions permitting the probe to bind to mRNA in the tissue sample; removing unbound probe from the sample; detecting the presence of bound, labelled probe in the sample; quantifying the amount of bound, labelled probe in the sample; and comparing the amount of bound, labelled probe in the sample to the amount of labelled probe which binds to a sample of non telencephalon-derived tissue or to a sample of telencephalon-derived tissue from a subject which expresses normal levels of the transcription factor in such tissue. Methods of isolating sample of telencephalon-derived tissue, of contacting such tissue with a labelled probe under conditions permitting binding of the probe to mRNA in the sample and of removing unbound probe from the sample are well known to those skilled in the art or can readily be determined by routine experimentation. Methods of detecting and quantifying the amount of bound, labelled probe in a sample are also well known to those skilled in the art. The transcription factor of this invention is not normally expressed in non telencephalon-derived tissue.

Identification of the transcription factor in such tissue is indicative of abnormal gene expression in the tissue. Comparison of the amount of bound, labelled probe in a sample of telencephalon-derived tissue from a subject animal with a similar sample from a normal animal will indicate whether the subject animal expresses an abnormal amount of the transcription factor of this invention. The presence of abnormal amounts of the transcription factor of this invention is related to the presence of an abnormal developmental state in the cells of telencephalon-derived tissue in which the transcription factor is expressed. The method provided by this invention is therefore useful in diagnosing the existence of an abnormal developmental state in the telencephalon derived tissue of an animal. Such animals include, but are not limited to mammals, e.g., mice, rats or humans.

This invention provides an expression vector comprising the isolated DNA molecule of this invention operably linked to a RNA polymerase promoter. Examples of such expression vectors include, but are not limited to, plasmids, viruses, cosmids or phages.

This invention provides a method of correcting an animal's defective synthesis of a transcription factor expressed in telencephalon-derived structures which comprises the steps of: isolating suitable cells from the animal; inserting the expression vector of this invention into the cells under conditions permitting stable integration of the vector into the genome of the cells and expression of the DNA molecule; and reintroducing the cells into the animal from which they were isolated. Cells suitable for use in accordance with the practice of this invention are well known to those skilled in the art or can readily be determined by routine experimentation. Methods of isolating such cells, and of readministering the cells to the animal, are also well known to those skilled in the art. "Inserting" an expression vectors as used herein is intended to mean any commonly accepted method of introducing an expression vector into cells such that the vector will stably integrate into the genome of the cell in a manner permitting the expression of the isolated DNA molecule in the integrated vector. Examples of such methods include, but are not limited to, calcium phosphate precipitation, electroporation or microinjection.

This invention also provides a method of correcting an animal's defective synthesis of a transcription factor expressed in telencephalon-derived structures which comprises the steps of: introducing the expression vector of this invention into a neuroepithelial cell line under conditions permitting stable integration of the vector into the genome of the neuroepithelial cells; and administering the neuroepithelial cells to the animal under conditions permitting the cells to grow in the animal and synthesize the transcription factor encoded by the expression vector. "Introducing" a vector into a cell in accordance with the practice of this invention is intended to mean any commonly accepted practice of introducing an expression vector into cells such that the vector will stably integrate into the genome of the cell in a manner permitting the expression of the isolated DNA molecule in the integrated vector. Examples of such methods include, but are not limited to, calcium phosphate precipitation, electroporation or microinjection. Neuroepithelial cells useful in accordance with the practice of this invention will be cells which can be administered to the animal without being rejected by the animal's immune system. Identification of such cells is within the purview of one of ordinary skill in the art. Methods of administering such cells are also well known to those skilled in the art.

This invention provides a host vector system comprising the expression vector of this invention in a suitable host cell. In one embodiment of this invention, the suitable host cell is a bacterial cell. In another embodiment of this invention, the suitable host cell is a eucaryotoic cell.

This invention also provides a method of producing a transcription factor expressed in the telencephalic region of the brian of a developing vertebrate animal or in structures derived therefrom, which comprises growing the host vector system of this invention under conditions which permit transcription and translation, followed by recovering the protein so produced. Conditions permitting transcription and translation in a host vector system are well known to one of ordinary skill in the art or can readily be determined by routine experimentation. Methods of recovering proteins expressed by a host vector system are also well known to those skilled in the art.

This invention provides an antibody which specifically recognizes the transcription factor of this invention. The antibody may be labelled with a detectable marker. In one embodiment of this invention, the antibody is a monoclonal antibody. This invention also provides a hybridoma cell which produces the monoclonal antibody of this invention. This invention further provides a method of producing a monoclonal antibody which specifically recognizes a transcription factor expressed in the telencephalic region of the brain of a developing animal or in structures derived therefrom which comprises culturing the hybridoma cell of this invention under conditions permitting antibody production, followed by recovering the antibody so produced. Methods of culturing hybridomas to produce monoclonal antibodies and of recovering monoclonal antibodies produced by hybridomas are also well known to those skilled in the art or can readily be determined by routine experimentation.

This invention provides a pharmaceutical composition comprising the antibody of this invention and a suitable carrier. Suitable carriers are well known to those of skill in the art or can readily be determined by hem without undue experimentation. Examples of suitable carriers include, but are not limited to, aqueous buffers. This invention also provides a method of detecting the presence in an animal of a transcription factor normally expressed in the telencephalic region of the brain of a developing animal or in structures derived therefrom which comprises the steps of: isolating a sample of suitable tissue from the animal; contacting the sample with the pharmaceutical composition of this invention, wherein the antibody is labelled with a detectable marker, under conditions permitting the antibody to bind to transcription factor in the sample; removing unbound, labelled antibody from the sample; and detecting the presence of bound antibody in the sample, said presence indicating the presence of transcription factor in the tissue sample. Methods of isolating telencephalon-derived tissue from an animal, of contacting the tissue with a pharmaceutical composition comprising a labelled antibody under conditions permitting binding of the antibody to protein in the sample and of removing unbound antibody from the sample and of detecting bound, labelled antibody in the sample are well known to those skilled in the art or can readily be determined by them without undue experimentation.

This invention also provides a method of detecting the presence in a patient of tumors arising from telencephalic-derived tissue which comprises the steps of: (i)isolating a sample of tumor tissue from the patient; contacting the sample with an effective amount of the pharmaceutical composition of claim 30, wherein the antibody is labelled with a detectable marker, under conditions permitting the antibody to bind to transcription factor in the sample; removing unbound, labelled antibody from the sample; and detecting the presence of bound, labelled antibody in the sample, said presence indicating the presence of transcription factor and thereby the presence of a tumor arising from telencephalic-derived tissue. Methods of isolating tumor tissue from an animal, of contacting the tissue with a pharmaceutical composition comprising a labelled antibody under conditions permitting binding of the antibody to protein in the sample and of removing unbound antibody from the sample and of detecting bound, labelled antibody in the sample are well known to those skilled in the art or can readily be determined by them without undue experimentation.

This invention will be better understood from the Examples which follow. However, those skilled in the art will readily appreciate that the specific examples detailed are only illustrative of the invention as described more fully in the claims which follow thereafter.

First Series of Experiments

Results

Isolation of cDNA clones for BP-1

We knew that Drosophila fork head or a closely related protein is expressed in the central nervous system of Drosophila (Weigel, Jurgens et al. 1989) while the three HNF-3 proteins were not expressed in the mammalian brain (Lai, Prezioso et al. 1991). Therefore we postulated that additional members of the HNF-3 gene family, which were critical to the development of the central nervous system, would be expressed in the mammalian brian. Northern analysis using an HNF-3a probe which spanned the DNA binding domain, was performed at low stringency. This revealed a 2.9 kb RNA species from whole brain tissue which was no longer detected after a high stringency wash. This probe was then used to screen a rat whole brain lambda gt11 DNA library. From a screen of 500,000 recombinants, 10 positive clones were obtained of which 6 proved to be from the same cDNA as assessed by restriction and partial sequence analysis. One clone was 2.9 kb in length and contained an open reading frame (FIGS. 1A-1C) which encodes a protein of 480 amino acids, which we call BF-1 (brain factor-1). The ATG at 443 is the translation initiation start site. It is the first to have a purine at the -3 position (Kozak 1986), is preceded by an in-frame termination codon and deletions of up to 331 nucleotides of the 5' end of the cDNA do not alter the size of the protein translated in reticulocyte lysates from RNA transcribed in vitro using T7 or T3 RNA polymerase compared to that translated from the full length cDNA (FIG. 2). The translated protein of 480 amino acids migrates with an apparent molecular weight on SDS-polyacrylamide gels of 67,000 daltons. For comparison, the translated HNF-3a protein of 466 amino acids migrates with an apparent size of 60,000 daltons. This size is different than previously reported (Lai et al. 1990) because the pre-stained molecular weight markers we have been using were recently recalibrated by the manufacturer (Sigma) to be larger than previously indicated. Both proteins migrate on SDS-gels more slowly than expected from the calculated size.

Expression of BF-1 is distinct from that of HNF-3α, 3β and 3γ

We examined the expression pattern of BF-1 initially by Northern analysis of poly A+ RNA isolated from both adult and fetal rat tissues. FIG. 3 shows that a 2.9 kB mRNA species is present in samples isolated from total brain using a probe derived from the BF-1 cDNA which does not contain the DNA binding domain. No expression is apparent in the lung, liver, and intestine (FIG. 3). The quantity of RNA in each lane is assessed by hybridization of the filter with a probe for GAPDH, which is found in similar abundance in all tissues. The Northern analysis was confirmed with RNase protection assays using a probe corresponding to nucleotides 1140-1449 of the cDNA. FIG. 4A shows the presence of the specifically protected probe of 309 nucleotides with total RNA from brain but not from liver, kidney, spleen and intestine. The quantity of the RNA was assessed to be equivalent among all the samples used in these assays by staining after electrophoresis in an agarose gel to visualize the 18S and 28S mRNA bands.

Levels of expression of BF-1 in the developing rat brain

Northern analysis showed that the mRNA was several-fold more abundant in embryonic day 17 (E17) brain than in the adult brain (FIG. 3). Additional studies which allowed better quantitation were performed with RNase protection assays on RNA from tissues during fetal development. FIG. 4 shows that BP-1 expression is detectable in E14 brain, increases to peak levels by day 17 and declines thereafter to adult levels. The data show that BF-1 is four-fold more abundant in E17 brain compared to the adult brain. Fetal and adult liver RNA were also assayed and not found to express BF-1 at any stage of development. We used total RNA isolated from dissected regions of the adult rat brain to study the regional distribution of BF-1 expression in RNAse protection assays. FIG. 4B reveals that BF-1 is expressed in the cortex, olfactory bulb, hippocampus and in the caudate putamen. Expression is absent in the cerebellum and thalamus. Adrenal tissue also does not express BF-1. The amount of RNA in each sample was verified to be the same in a Northern blot using a probe to 18S RNA (Ehrlich et al. 1990).

Expression of IF-1 is restricted to the telencephalon of the developing brain

Examination of rat brain sections by in situ hybridization, confirmed the region restricted expression pattern of BF-1. FIGS. 5J and 5K are autoradiographs of sections of adult brain hybridized with an antisense probe for BF-1. The cerebral cortex, caudate putamen, hippocampus and dentate gyrus show levels of BF-1 expression significantly above background while the thalamus, cerebellum and brainstem do not. This restricted expression to telencephalon-derived structures is even more clearly seen in the E17 brain. FIG. 5E shows a brightfield view of a parasagittal brain section. The autoradiograph of this section (FIG. 5F) and two coronal sections (FIGS. 5G and H) shows high level expression in the cerebral cortex and caudate putamen and no significant expression in the more caudal regions of the brain. The specificity of this signal for BF-1 is shown in FIG. 5I, an autoradiograph of a section parallel to that shown in 5E, hybridized with a sense probe to BF-1. This restriction of BF-1 expression is established early in development. FIGS. 5A-D show that BF-1 expression is limited in the nervous system to the most rostral region of the developing brain. We detect high levels of BF-1 MRNA in the ventricular zone surrounding the lateral ventricles at E11.5 soon after the telencephalic evaginations arise (Rugh 1968). The level of expression declines between E17 and the adult (FIGS. 5J,K), consistent with the results obtained with analysis of whole brain RNA. The autoradiographs for the embryonic sections are exposed for one-third of the time as the adult sections. Thus expression levels per cell falls about 10-20 fold.

Higher magnification views show more clearly the restriction of expression within the developing forebrain to the telencephalon. High levels of BF-1 are detectable in the rostral neuroepithelium of E10 embryos, shown in matching brightfield and darkfield views (FIGS. 6A-6D). At this stage, it is difficult to distinguish the border between the telencephalic and diencephalic neuroepithelium. The rapid proliferation of the cells of the telencephalon results in distinct telencephalic vesicles by E11.5 (FIG. 6C). Here we obverse high levels of BF-1 expression in the neuroepithelium surrounding the lateral ventricles. By contrast, structures of the diencephalic neuroepithelium, including the optic stalk and the optic cup which will develop into the retina, are clearly devoid of BF-1 (FIGS. 6C, 5A, 5B). In order to further demonstrate the restriction of BF-1 expression to the telencephalic neuroepithelium and the structures arising from it, we show two additional views of the telencephalon and diencephalon from E13.5 in FIG. 7. At this stage, the earlier onset of neurogenesis in the basal telencephalon is apparent by the presence of larger differentiating fields around the basal telencephalic neuroepithelium, compared with the cerebral cortical neuroepithelium (FIGS. 7A and 7B). The differentiating cells of the basal telencephalon express BF-1 at least as highly as the neuroepithelium. Expression of BF-1 abruptly terminates caudal to this region. An adjacent section further shows the absence of expression in the diencephalon and also shows absence of expression in the medial wall of the telencephalic vesicles (FIGS. 7C and 7D). This region of the telencephalic neuroepithelium expresses BF-1 earlier in development but begins to decrease expression with the infolding of the superior bridge and the establishment of the telencephalic vesicles (FIGS. 6E and 6F).

A few localized sites of expression are detected outside of the central nervous system, including the olfactory placode which gives rise to the nasal epithelium (FIG. 6C), the auditory vesicle and the pharyngeal pouches (FIGS. 6C and 6D). Expression at these sites declines through embryogenesis. The absence of expression through development in the tissues where HNF-3 proteins are expressed, the liver, the lung and the intestine are confirmed by in-situ data (FIGS. 5A-D).

BF-1 shares homology with the HNF-3 family in the DNA binding domain

Comparison of the sequence of BF-1 with the members of the HNF-3 family shows that it contains a homologous region from amino acids 162-271 corresponding to the DNA binding domain previously identified in the HNF-3 family (FIGS. 1A-1C). However, whereas the HNF-3 family members are 85% identical within this entire 110 amino acid region, BF-1 is considerably more divergent. This divergence is greater than that of the Drosophila fork head gene from the rat HNF-3 genes. Thus, fork head is the Drosophila homolog of HNF-3α, 3β and 3γ, while BF-1 is in a distinct subfamily. The homology is not uniform within this domain. It is apparent that the central portion with 74% of the amino acids (32/43 aa) identical comprises the most highly conserved structure while the carboxyl third of this region is only 36% identical (13/36 aa). FIG. 1 shows the amino acids in the DNA binding domain which are identical to at least two of the other four members of the HNF-3/fork head family. These differences raised the likelihood that BF-1, if it bound to DNA, might have a distinct binding specificity. The sequence of BF-1 is notable for several other features. First, it does not share the two other conserved short domains II and III carboxyl to the binding domain which are found in the HNF-3 and fork head genes (Lai, Prezioso et al. 1991). However, a short sequence similar to that in domain II is present in the amino terminal end of BF-1 (amino acids 18-24, FIG. 1). The function of these domains remains unknown. Finally, BF-1 contains proline and glutamine rich regions, which have previously been shown in other transcription factors to confer activation function (Courey and Tjian 1988; Mermod et al. 1989).

BF-1 is a sequence specific DNA binding protein with a distinct binding specificity

Because of the differences in the amino acid sequence in the binding domain as compared with HNF-3 proteins, we were not surprised to find that BF-1 did not bind readily to the strong HNF-3 site from the transthyretin (TTR) promoter. This site has been previously shown to be located between nucleotides -85 and -111, upstream of the transcription initiation site (Costa et al. 1989). However, when we used a large amount of bacterially expressed fusion protein (25 ng) which contained the binding domain of BF-1, we were able to detect a weak complex in gel mobility shift experiments using this probe. This suggested that BF-1 was a DNA binding protein with a low affinity for this site. We reasoned that a preferred binding site would have some similarity to the site in the TTR promoter, so we obtained and tested a series of oligonucleotides which contained different sites which bind to the HNF-3 proteins. We compared two bacterially produced fusion proteins containing 206 amino acids of the BF-1 protein in gel mobility shift assays with HNF-3 proteins from a liver extract. Interestingly, we found one HNF-3 site from the promoter of the HNF-1a gene called B2 (Kuo et al. 1992) which binds to BF-1 with at least twenty-fold greater affinity than any of four other sites tested. FIG. 8 shows a gel mobility shift experiment using two different probes, the TTR promoter site and the B2 site. Whereas liver nuclear extract (containing at least three HNF-3 proteins) binds to the TTR site with a slightly higher affinity compared to the B2 site, both BF-1 fusion proteins have a markedly greater affinity for the B2 site compared to the TTR site. One fusion protein using the pET vector adds 14 aa to the amino terminal end (Rosenberg et al. 1987) while the other using the pGEX vector adds 27.5 kD of glutathione tranferase to the amino terminal end (Smith and Johnson 1988). These 206 amino acids of BF-1 contain the entire DNA binding domain as determined by sequence homology with the HNF-3 proteins. It has been determined for HNF-3 proteins that the binding domain alone has DNA binding properties indistinguishable from the intact protein (Tao and Lai, unpublished data). BF-1 also did not bind to three other HFN-3 sites from the α-1 antitrypsin gene, a second site from the transthyretin gene (Costa, Grayson et al. 1989), and the albumin gene (Liu et al. 1991). These results show that BF-1 has a distinct DNA binding specificity from that of the HNF-3 proteins. The identical DNA binding properties of two different BF-1 fusion proteins indicate that DNA binding is attributable to the BF-1 portion of the protein. However, the affinity of BF-1 for the B2 site is relatively low compared to the affinity of the HNF-3 proteins for this site. Studies are in progress to identify the optimal recognition sequences for this protein.

Discussion

We have cloned and characterized, BF-1, a new member of the HNF-3 family from a rat brain cDNA library. BF-1 shares homology in the DNA binding domain with HNF-3α, 3β, 3γ and Fork head but clearly belongs to a different subfamily based on significant divergence in the carboxyl third of this domain. This divergence in sequence likely reflects some differences in function as underscored by our findings that BF-1 is expressed in a completely distinct pattern than the HNF-3 proteins and that BF-1 has a different DNA binding specificity. Consistent with a role in central nervous system development, BF-1 is expressed at high levels in specific regions of the brain during embryogenesis. These results suggest that an extended family of transcription factors related to the HNF-3 proteins exists which have a boarder role in mammalian development than previously believed. Members of the extended HNF-3 family are not only expressed in gut endoderm derived tissues but in the central nervous system as well. The emerging picture is that of a large gene gamily with distinct subfamilies of transcription factors, each of which may regulate the development of distinct subsets of tissues. The brain-specific subfamily comprises BF-1 and at least two additional members. An additional member of this family which is present in lymphoid cells has recently been reported (Li et al. 1991).

The restricted expression of BF-1 to the telencephalon at early stages in brain development suggests that it plays a role in the regional differentiation of the neural tube. It has been suggested that signals from the underlying mesoderm during gastrulation lead to the commitment of different regions of the neural tube to form different parts of the central nervous system. The forebrain forms above the prechordal plate (Bergquist and Kallen 1954), rather than the notochord and floor plate which has been shown to provide inductive signals to the spinal cord (Placzek, Tessier-Lavigne et al. 1990; Yamada et al. 1991). Thus, it would be expected that different signals are received in this region of the neural tube, resulting in distinct patterns of gene expression. The forebrain subsequently subdivides into the telencephalon and the diencephalon. The restricted expression of BF-1 to the rostral portion of the forebrain prior to its morphologically apparent subdivision into two vesicles and its structural homology to fork head, a putative transcriptional regulator, suggests that it is likely to be an important intermediary in the regulatory cascade which commits the cells of the rostral neural tube to the formation of the telencephalon. The fork head mutation causes a homeotic transformation of the terminal regions of the embryo. Foregut and hindgut are replaced by ectopic head structures (Jurgens and Weigel 1988). BF-1 expression is first evident in the proliferating neuroepithelial precursor cells surrounding the telocoel. As development progresses, these cells differentiate into neuronal and glial cells and migrate outward. The expression of BF-1 persists in the post-mitotic cells and by E17 is uniformly detected in the telencephalic structures.

Expression is clearly evident in neurons as exemplified by the in-situ signals of the hippocampus and dentate gyrus (FIG. 5K). Expression in the adult is restricted to the gray matter and the large increase in BF-1 expression between E14 and E17 correlates well with the period of rapid neuronal proliferation. Furthermore, expression of BF-1 per cell in the cerebral hemispheres declines postnatally, correlating with the major period of glial proliferation. These results suggest that BF-1 is primarily expressed in neurons but we cannot exclude expression in glia. The relative uniformity of expression in the cerebral cortex and the caudate putamen indicates that BF-1 does not primarily direct the final differentiation of the many neuronal phenotypes found in these parts of the brain. Rather, BF-1 appears to function at an earlier step in the differentiation pathway to commit the precursor cells to form specific regions of the brain and not specific cell types. Its restriction to the rostral end of the neural tube is reminiscent of the restriction of fork head to the anterior and posterior termini of the early Drosophila embryo. Thus we postulate that BF-1 functions in regional specialization of the central nervous system in a manner analogous to the function of fork head in the regional specialization of the terminal structures in Drosophila.

Other putative transcriptional regulators have been found to be restricted to specific regions of the developing brian, including several homeodomain proteins and one zinc finger protein in specific rhombomeres of the hindbrain (Murphy, Davidson et al. 1989; Wilkinson, Bhatt et al. 1989; Wilkinson, Bhatt et al. 1989; Kessel and Gruss 1990; He and Rosenfeld 1991). Only a few examples of region-specific expression exists for the other regions of the brain. En-2 is expressed at the midbrain and hindbrain junction(Davis and Joyner 1988; Davis et al. 1988) and recently, two genes related to Distal-less were found to be expressed in a restricted pattern within the forebrain. Tes-1 and Dlx are expressed in the ventral forebrain of the mid-gestation embryo, in both the telencephalon and diencephalon (Porteus et al. 1991; Price et al. 1991). These factors along with BF-1 are likely to be part of the molecular basis of the regional organization of the developing neural tube. Still other transcription factors such as several members of the POU-domain family which are expressed in specific patterns in the developing brain but are not restricted to any single region (He et al. 1989), may function to determine specific phenotypes such as the different types of neurons and glia. The function of BF-1 in the adult brain is not readily apparent but may be related to maintaining the expression of those genes which are common to telecephalon derived structures. The product of one such gene has been detected as a telencephalon specific antigen in the rabbit (Mori et al. 1987) This antigen is restricted to the gray matter of the cerebral hemispheres and increases postnatally from low levels at birth.

Experimental Procedures:

Isolation and sequencing of cDNA clones of rat and murine BF-1

The rat BF-1 cDNA was obtained by screening a lgt11 library from whole adult rat brain generously provided by Dr. Streamsen Chua, using a random hexamer primed probe spanning nucleotides 596-923 of the HNF-3α cDNA. This probe contains the region encoding the DNA binding domain. Hybridization and washes were performed as previously described except for hybridization at 50° C. and the final wash at 50° C. in 2× SSPE. From 500,000 recombinants, 10 positive clones were analyzed further. Six of these proved to be overlapping clones of a single cDNA as determined by restriction mapping and partial sequence. The largest clone was used for subsequent studies.

Sequencing was performed by the dideoxy chain termination method on double stranded templates. The sequence was determined completely on both strands with overlapping subcloned and exonuclease shortened templates, and for nucleotides 1-2000 also with ITP-containing sequencing mixes (United States Biochemicals Sequenase kit) to eliminate compression artifacts.

Northern and ribonuclease protection analyses

RNA was isolated from rat tissues by the acid guanidinium thiocyanate-phenol-chloroform extraction method (Chomczynski and Sacchi 1987; Puissant and Houdebine 1990). Pregnant Sprague-Dawley rats were obtained from Harlan Sprague Dawley, timed at embryonic day 0.5 on the morning following an evening mating. Poly(A)+RNA was selected by chromatography on oligo(dT) cellulose. Northern analyses with 2 mg poly-A+ RNA were performed as previously described.

Ribonuclease protection assays were performed with 10 mg total RNA as described previously (Costa et al. 1986). RNA probes were synthesized from a pBluescript plasmid containing the fragment of the BF-1 cDNA from nucleotides 1140-1149, linearized with PvuII, using T7 polymerase. The probe is 457 nucleotides in length and the protected fragment is 309 nucleotides. Samples were analyzed on 6% acrylamide-urea gels which were dried and exposed to X-ray film with screens for 16-48 hrs.

In-situ hybridization

Brain tissue was obtained from adult male Sprague-Dawley rats perfused with 4% paraformaldehyde or rat embryos. 8 micron paraffin sections were dewaxed in xylene and then processed essentially as described previously (Kuo et al. 1988). S-35 labeled riboprobes were synthesized using T7 (antisense) or T3 (sense) RNA polymerase from linearized plasmid templates containing the BF-1 cDNA from the Nhe I site at nucleotide 1265 to the 3' end. This probe does not contain the sequence encoding the DNA binding domain. Hybridization was for 16 hrs at 58° C. and the high stringency washes were performed at 60° C. in 2× SSC/50% formamide. Embryonic tissue sections were exposed to film for 16 hrs and to NTB-2 emulsion for 48 hrs. Adult sections were exposed for 48 hrs and 6 days respectively.

Fusion proteins and gel mobility shift assays

The fragment of the rat BF-1 cDNA from nucleotides 785 to 2240 was cloned into the BamHI site of the pGEX-3X vector (Pharmacia®) which yields a protein with 27.5K of glutathione transferase fused to 206 amino acids of BF-1 containing the DNA binding domain. The protein was expressed in E. coli SF8 cells. The fusion protein was purified from the crude bacterial extract by adsorption to glutathione agarose beads followed by elution with glutathione (Smith and Johnson 1988). The quantity of the partially purified protein used in binding assays was determined by Coomassie Blue staining. A second expression plasmid was constructed by ligating this fragment of BF-1 into the BamHI site of the pET-3c vector which yields a protein of 14 amino acids fused to 206 amino acids of BF-1. The crude lysate from bacteria expressing this fusion protein were used directly in binding assays (Studier and Moffatt 1986). 5 μg of BF-1 pET bacterial lysate, 25 ng of BF-1 pGEX, or 5 μg of liver nuclear extract was incubated with either B2 or TTR oligonucleotide probe as described previously (Lai, Prezioso et al. 1990).

The sequence of the binding site oligonucleotide from the transthyretin (TTR) promoter and the gel mobility shift assay have been described previously. The sequence of the binding site oligonucleotide from the HNF-1 promoter (B2) has also been described (Kuo, Conley et al. 1992).

Second Series of Experiments

Cloning of BF-1 cDNA into plasmid pBluescript KS

Lambda phage carrying the rat BF-1 cDNA was purified and the phage DNA was isolated by standard methods.

cDNA in lambda gt11 vector was excised with restriction enzymes EcoRI and NotI and cloned into the plasmid vector pBluescript KS.

This cDNA were cloned into EcoRI and NotI sites of pBluescript KS plasmid vector and can be excised using these restriction enzymes (FIG. 12). The plasmid made is designated, pBSKS-BF-1.

Mouse BF-1 cDNA

Several cDNA from a new born mouse brain lambda ZAPII library (purchased from Stratagene) was isolated using a probe from the rat BF-1 cDNA. Plasmid containing the cDNA were excised from the lambda phage according to the protocols of Stratagene. These plasmids contain the cDNA was EcoRI and XhoI fragments. Three distinct cDNA were identified based on sequence comparison. One is completely homologous to the rat cDNA and is >98% identical (mN13). A second cDNA is homologous except at the 5' end which diverges completely (mN3). A third has a deletion in the 3' untranslated region (mN11). These three cDNA arise from the alternative splicing of transcripts from one gene.

Genomic DNA for BF-1

The rat cDNA for BF-1 was used as a probe to screen and isolate a lambda phage clone containing the mouse genomic DNA for BF-1. The vector is lambda Gem 12 (Promega) and the insert can be excised with several restriction enzymes including BamHI. A clone was identified which contains an approximately 15 kB piece of genomic DNA which includes the entire sequence encoding the BF-1 protein, as well as approximately 10 kB of 5' flanking sequence. The 3' end of the clone is not yet fully mapped, but contains the 3' flanking sequence. These flanking sequences contain the regulatory elements which control the expression of BF-1 in a cell-specific manner. We have identified the transcription start sites for BF-1. There are two major sites and several minor sites. A restriction map of the genomic DNA is shown in FIG. 9 with the positions of the major start sites. The two major start sites leads to the two distinct cDNA which are divergent at the 5' end (mN3 and mN13). The protein translated from these two cDNA are identical.

Generation of a vector useful to express protein of interest in a cell-specific manner

The nontranscribed regions of the BF-1 gene was further studied.

Approximately 10 kilobase of the 5' end of the nontranscribed region and about 3 kilobase of the 3' end of the nontranscribed region of the BF-1 gene are subcloned in a plasmid, called pTBL3. A lacZ gene was inserted between the 5' and 3' regions to confirm and define the regulatory regions which are necessary to define the expression pattern of this gene. Using this plasmid, transgenic mouse is made.

Map of the Bf-1-lacZ expression cassette in plasmid pTBL3. Sfil and Xhol linkers were introduced into the Sphl and EcoRI sites of the vector, pGEM4 respectively. Then whole BF-1-lacZ expression cassette was cloned into the Sfil and Xhol sites of the modified vector. Ncol and Kpnl are not unique sites.

Generation of transgenic mice

The linearized DNA fragment containing the BF-1 gene and lacZ sequence was purified and injected into mouse eggs. These eggs are then reimplanted into the reproductive tract of female mice and allowed to develop to term according to standard protocols.

The lacZ expression in the transgenic animal reflects the expression pattern regulated by the BF-1 gene. Expression is monitored by staining the tissues from the animal in a solution which yields a blue color in the cells which express β-galactosidase activity encoded by lacZ. One line of mice we have generated demonstrates restriction of lac Z expression to the telencephalic structures of the brain.

Analysis of the transgenic mouse generated indicates the lac-z gene is now expressing in the cell which expresses the BF-1 gene. This result show that the non transcribing regions is useful to direct expression of a protein of interest in a cell-specific manner in the brain.

Third Series of Experiments

Molecular cloning of BF-2 (Brain Factor-2) and BF-3 (Brain Factor-3)

Rat BF-2 and BF-3 partial cDNA Using the HNF-3a cDNA probe used for isolating BF-1 cDNA, we also isolated two additional cDNA which are distinct but related to BF-1 by sequence homology. These cDNA were isolated from lambda gt11 with inserts cloned into EcoRI and NotI sites. These cDNA contain only part of the protein coding sequence for these two proteins. These cDNA were cloned into EcoRI and NotI sites of pBluescript KS.

The rat BF-2 cDNA was cloned into EcoRI and NotI sites of pBluescript KS plasmid vector and can be excised using these restriction enzymes. The plasmid made is designated, pBSKS-BF-2.

The rat BF-3 cDNA was cloned into EcoRI and NotI sites of pBluescript KS plasmid vector and can be excised using these restriction enzymes. The plasmid made is designated, BF-3.

Mouse full length cDNA for BF-2 and BF-3

Using the corresponding rat cDNA probes, the mouse homologs for BF-2 and BF-3 were isolated from cDNA libraries. The BF-2 cDNA was isolated from a new born mouse brain lambda ZAPII library (purchased from Stragene) and the BF-3 cDNA was isolated from a lambda shlox library from mouse 13 day embryos (purchased from Novagen).

A fragment of rat cDNA was labelled with P-32 and used to screen lambda phage DNA transferred to nitrocellulose membranes. Hybridization and washes were performed as described for the isolation of rat BF-1.

Sequences of the protein coding region of BF-2 are shown in FIGS. 10A and 10B (Seq. I.D. No.3). FIG. 11 (Seq.I.D. No.4) shows a partial sequence of BF-3.

Properties of BF-2 and BF-3

BF-2 is a transcription factor which is expressed in the diencephalon of the central nervous system. It is also expressed in several other sites outside of the nervous system. BF-3 is expressed in the brain and its expression is limited (not ubiquitously expressed).

Human BF-1, BF-2 and BF-3

Human cDNA corresponding to BF-1, BF-2 and BF-3 can be readily obtained by screening a human brain cDNA library using the probes we used to isolate the mouse cDNA. The sequence similarity between rat and mouse cDNA of greater than 95% for all three of these cDNA, implying that the human sequence will also be highly homologous and readily obtainable by these methods.

References:

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    __________________________________________________________________________     #             SEQUENCE LISTING                                                    - -  - - (1) GENERAL INFORMATION:                                              - -    (iii) NUMBER OF SEQUENCES: 4                                            - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                      - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 2830 base - #pairs                                                 (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: cDNA                                               - -    (iii) HYPOTHETICAL: N                                                   - -     (iv) ANTI-SENSE: N                                                     - -     (ix) FEATURE:                                                                   (A) NAME/KEY: CDS                                                              (B) LOCATION: 443..1882                                                        (D) OTHER INFORMATION:                                                - -     (ix) FEATURE:                                                                   (A) NAME/KEY: misc.sub.-- - #feature                                           (B) LOCATION: 926..1255                                                        (D) OTHER INFORMATION: - #/note= "nucleotide sequence                              encoding - #DNA binding domain                                                 homology"                                                        - -     (ix) FEATURE:                                                                   (A) NAME/KEY: misc.sub.-- - #signal                                            (B) LOCATION: 1883..1885                                                       (D) OTHER INFORMATION: - #/note= "translation termination                           codon"                                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                - - GGCGGCCGCT CCGGGACGCG CCCGCGCGCT GCCCGGCTCT CCCCCCCTTC GG -              #GCTGCCGC     60                                                                  - - TGCTGCTGCT GTGACTGCTG CGGCGCGAGG AGGAGGAGGC AGCGGGGGAG GG -             #GGAGGCCG    120                                                                  - - GGCGCGGAAC GGAGCGGGGC GCTGCACCCC GGGCGACGGG TTGCTTCTGC CT -             #CTAGCTTC    180                                                                  - - TCTCTCTCTC TCTCTCTCTC TCTCTCTCTC TCTCTCTCTC TCTCTCTCTC TC -             #TCTCTCTC    240                                                                  - - TAATTCTTGA GGGGTGGTTG CAGCTTTTGC TACATGCCTT GCCAGCGCCG GA -             #GCCTGCGG    300                                                                  - - TCCAACTGCG CTGCTGCCGG AGCGCTCAGT GCCGCCCCCG CTGCCCGCTC CC -             #CCCGCTCC    360                                                                  - - CCACTCCGAA CCCGCCGGTC GCTCGCCGCG CTGCTGCTCG GCTCCTGCGC CG -             #CCGCCGTC    420                                                                  - - GCCCCCCCCC GACGCCTGGG TG ATG CTG GAC ATG GGA GAT - # AGG AAA GAG        GTG     472                                                                                        - #       Met Leu Asp Met Gly Asp A - #rg Lys Glu Val                         - #         1         - #      5            - #             10                                                                                - - AAA ATG ATT CCC AAG TCC TCG TTC AGC ATC AA - #C AGC CTG GTC CCT         GAG      520                                                                     Lys Met Ile Pro Lys Ser Ser Phe Ser Ile As - #n Ser Leu Val Pro Glu                            15 - #                 20 - #                 25               - - GCC GTC CAG AAC GAC AAC CAC CAC GCG AGC CA - #C GGC TAC CAC AAC AGC           568                                                                        Ala Val Gln Asn Asp Asn His His Ala Ser Hi - #s Gly Tyr His Asn Ser                         30     - #             35     - #             40                   - - CAC CAC CCC CAG CAT CAC CAT CAT CAT CAC CA - #C CAC CAC CAC CCG CCG           616                                                                        His His Pro Gln His His His His His His Hi - #s His His His Pro Pro                     45         - #         50         - #         55                       - - CCG CCC GCG CCT CAG CCG CCG CCA CCG CCG CC - #C CAG CAG CAG CAG CAG           664                                                                        Pro Pro Ala Pro Gln Pro Pro Pro Pro Pro Pr - #o Gln Gln Gln Gln Gln                 60             - #     65             - #     70                           - - CCG CCC CCG GCC CCG CAG CCC CCG CAG GCG CG - #C GGC GCC CCA GCA GCG           712                                                                        Pro Pro Pro Ala Pro Gln Pro Pro Gln Ala Ar - #g Gly Ala Pro Ala Ala             75                 - # 80                 - # 85                 - # 90        - - GAC GAC GAC AAG GGC CCC CAG CCG CTT CTG CT - #C CCG CCG TCC GCC GCC           760                                                                        Asp Asp Asp Lys Gly Pro Gln Pro Leu Leu Le - #u Pro Pro Ser Ala Ala                             95 - #                100 - #                105               - - CTG GAC GGG GCC AAG GCT GAC GCA CTT GGA GC - #C AAA GGC GAG CCA GGC           808                                                                        Leu Asp Gly Ala Lys Ala Asp Ala Leu Gly Al - #a Lys Gly Glu Pro Gly                        110      - #           115      - #           120                   - - GGC GGG CCT GCG GAG CTG GCG CCC GTC GGG CC - #G GAC GAG AAG GAG AAG           856                                                                        Gly Gly Pro Ala Glu Leu Ala Pro Val Gly Pr - #o Asp Glu Lys Glu Lys                    125          - #       130          - #       135                       - - GGC GCG GGC GCT GGG GGG GAG GAG AAG AAG GG - #G GCG GGC GAG GGC GGC           904                                                                        Gly Ala Gly Ala Gly Gly Glu Glu Lys Lys Gl - #y Ala Gly Glu Gly Gly                140              - #   145              - #   150                           - - AAG GAC GGG GAG GGG GGC AAG GAG GGC GAC AA - #G AAC AAC GGC AAG TAC           952                                                                        Lys Asp Gly Glu Gly Gly Lys Glu Gly Asp Ly - #s Asn Asn Gly Lys Tyr            155                 1 - #60                 1 - #65                 1 -       #70                                                                               - - GAG AAG CCG CCG TTC ACC TAC AAC GCG CTC AT - #C ATG ATG GCC ATC         AGG     1000                                                                     Glu Lys Pro Pro Phe Thr Tyr Asn Ala Leu Il - #e Met Met Ala Ile Arg                           175  - #               180  - #               185               - - CAG AGT CCC GAG AAG CGC CTG ACG CTC AAC GG - #C ATC TAC GAG TTC ATC          1048                                                                        Gln Ser Pro Glu Lys Arg Leu Thr Leu Asn Gl - #y Ile Tyr Glu Phe Ile                        190      - #           195      - #           200                   - - ATG AAG AAC TTC CCT TAC TAC CGC GAG AAC AA - #G CAG GGC TGG CAG AAC          1096                                                                        Met Lys Asn Phe Pro Tyr Tyr Arg Glu Asn Ly - #s Gln Gly Trp Gln Asn                    205          - #       210          - #       215                       - - TCC ATC CGC CAC AAC CTG TCC CTC AAC AAG TG - #C TTC GTG AAG GTA CCG          1144                                                                        Ser Ile Arg His Asn Leu Ser Leu Asn Lys Cy - #s Phe Val Lys Val Pro                220              - #   225              - #   230                           - - CGC CAC TAC GAC GAC CCG GGC AAG GGC AAC TA - #C TGG ATG CTG GAC CCG          1192                                                                        Arg His Tyr Asp Asp Pro Gly Lys Gly Asn Ty - #r Trp Met Leu Asp Pro            235                 2 - #40                 2 - #45                 2 -       #50                                                                               - - TCG AGC GAC GAC GTG TTC ATC GGC GGC ACG AC - #C GGC AAG CTG CGG         CGC     1240                                                                     Ser Ser Asp Asp Val Phe Ile Gly Gly Thr Th - #r Gly Lys Leu Arg Arg                           255  - #               260  - #               265               - - CGC TCC ACC ACG TCT CGG GCC AAG CTA GCC TT - #T AAG CGC CGG GCA CGG          1288                                                                        Arg Ser Thr Thr Ser Arg Ala Lys Leu Ala Ph - #e Lys Arg Arg Ala Arg                        270      - #           275      - #           280                   - - CTC ACC TCC ACC GGC CTC ACC TTC ATG GAC CG - #C GCC GGC TCC CTC TAC          1336                                                                        Leu Thr Ser Thr Gly Leu Thr Phe Met Asp Ar - #g Ala Gly Ser Leu Tyr                    285          - #       290          - #       295                       - - TGG CCC ATG TCG CCC TTC CTG TCC CTG CAC CA - #C CCT CGC GCC AGC AGC          1384                                                                        Trp Pro Met Ser Pro Phe Leu Ser Leu His Hi - #s Pro Arg Ala Ser Ser                300              - #   305              - #   310                           - - ACT TTG AGT TAC AAC GGG ACC ACC TCG GCC TA - #C CCC AGC CAC CCC ATG          1432                                                                        Thr Leu Ser Tyr Asn Gly Thr Thr Ser Ala Ty - #r Pro Ser His Pro Met            315                 3 - #20                 3 - #25                 3 -       #30                                                                               - - CCC TAC AGC TCC GTG TTG ACT CAA AAC TCG CT - #G GGC AAC AAC CAC         TCC     1480                                                                     Pro Tyr Ser Ser Val Leu Thr Gln Asn Ser Le - #u Gly Asn Asn His Ser                           335  - #               340  - #               345               - - TTC TCC ACC GCC AAC GGG CTG AGC GTG GAC CG - #G CTG GTC AAC GGG GAG          1528                                                                        Phe Ser Thr Ala Asn Gly Leu Ser Val Asp Ar - #g Leu Val Asn Gly Glu                        350      - #           355      - #           360                   - - ATC CCG TAC GCC ACG CAC CAC CTC ACG GCC GC - #T GCG CTC GCC GCC TCC          1576                                                                        Ile Pro Tyr Ala Thr His His Leu Thr Ala Al - #a Ala Leu Ala Ala Ser                    365          - #       370          - #       375                       - - GTG CCC TGC GGC CTG TCG GTG CCC TGC TCC GG - #G ACC TAC TCC CTC AAC          1624                                                                        Val Pro Cys Gly Leu Ser Val Pro Cys Ser Gl - #y Thr Tyr Ser Leu Asn                380              - #   385              - #   390                           - - CCC TGC TCC GTC AAC CTG CTC GCG GGC CAG AC - #C AGT TAC TTT TTC CCC          1672                                                                        Pro Cys Ser Val Asn Leu Leu Ala Gly Gln Th - #r Ser Tyr Phe Phe Pro            395                 4 - #00                 4 - #05                 4 -       #10                                                                               - - CAC GTC CCG CAC CCG TCA ATG ACT TCG CAG AC - #C AGC ACG TCC ATG         AGC     1720                                                                     His Val Pro His Pro Ser Met Thr Ser Gln Th - #r Ser Thr Ser Met Ser                           415  - #               420  - #               425               - - GCC CGG GCC GCG TCC TCC TCT ACG TCG CCG CA - #G GCC CCC TCG ACC CTG          1768                                                                        Ala Arg Ala Ala Ser Ser Ser Thr Ser Pro Gl - #n Ala Pro Ser Thr Leu                        430      - #           435      - #           440                   - - CCC TGT GAG TCT TTA AGA CCC TCT TTG CCA AG - #T TTT ACG ACA GGA CTG          1816                                                                        Pro Cys Glu Ser Leu Arg Pro Ser Leu Pro Se - #r Phe Thr Thr Gly Leu                    445          - #       450          - #       455                       - - TCC GGG GGA CTG TCT GAT TAT TTC ACA CAT CA - #A AAT CAG GGG TCT TCT          1864                                                                        Ser Gly Gly Leu Ser Asp Tyr Phe Thr His Gl - #n Asn Gln Gly Ser Ser                460              - #   465              - #   470                           - - TCC AAC CCT TTA ATA CAT TAACATCCCG GGGACCAGAC TG - #TAAGTGAA                 1912                                                                        Ser Asn Pro Leu Ile His                                                        475                 4 - #80                                                     - - CGTTTTACAC ACATTTGCAT TGTAAATGAT AATTAAAAAA TAAGTCCAGG TA -              #TTTTTTAT   1972                                                                  - - TAAGCCCTTC CCCCCATTTC TGTACGTTTG TTCAGTCTTT AGGGTTGTTT AC -             #TATTCTAA   2032                                                                  - - CACGGTGTGG AGTGTCAGCA GCGAGGTGCA ATGTGGGAGA ATACATTGTA GA -             #ATATAAGG   2092                                                                  - - TTTGGACGTC AAATTATAGT AGAATGTGTA TCTAAATAGT GACTGCTTTG CC -             #ATTTCATT   2152                                                                  - - CAAACCTGAC AAGTCTATCT CTAAAGGCTG CCAGATTTCC ATGTGTGCAG TA -             #TTATAAGT   2212                                                                  - - TATCATGGAT CTATCTGGTG GACGCAGGCC TTGAAGAACA ACCTAAATTA TG -             #AAGAGAGT   2272                                                                  - - TTTAAAATGT TAAACTGTAA TTTGAATGTA AGAATTTGTA GGTAAAGGTG CC -             #CAAGAAAT   2332                                                                  - - TATATTGGCC ATTTATTGTT TTGTCCTTTT CTTTAAAGAA CTGTTTTTTT CT -             #TTTGTTTA   2392                                                                  - - CTTTTAGACC AAAGATTGGA TTCTAGCAAA TGCACTTGGT ATACTAAGTA TT -             #AAAACAAG   2452                                                                  - - CAAACAAACA AACAAAAAAA GGAAGGTTGT TTAGTTTGGC AACACTGCCC AT -             #TCAATTGA   2512                                                                  - - ATCCGAAAGG ACAAAATTAA GGATTGCCTT CAGTTTGTGT TGTGTATATT TC -             #GATGTATG   2572                                                                  - - TGGTCACTAA CAGGTCACTT TATTTTTTCT AAATGTAGTG AAATGTTAAT AC -             #CTATTGTA   2632                                                                  - - CTTATAGGTA AACCTTGCAA ATATGTAACC TGTGTTGCGC AAATGCCGCA TC -             #AATTTGAG   2692                                                                  - - TGATTGTTAA TGTTGCTTAA AAATTCTTGA TTGTGATACT GTGGTCATAT GC -             #CCTTGTTT   2752                                                                  - - GTCACTTACA AAAATGTTTA CTATGAACAC ACAGAAATAA AAAATAGGCT AA -             #ATTCATAT   2812                                                                  - - ATAAAAAAAA AAAAAAAA             - #                  - #                       - #2830                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                      - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 480 amino - #acids                                                 (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: protein                                            - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                - - Met Leu Asp Met Gly Asp Arg Lys Glu Val Ly - #s Met Ile Pro Lys Ser         1               5 - #                 10 - #                 15               - - Ser Phe Ser Ile Asn Ser Leu Val Pro Glu Al - #a Val Gln Asn Asp Asn                    20     - #             25     - #             30                   - - His His Ala Ser His Gly Tyr His Asn Ser Hi - #s His Pro Gln His His                35         - #         40         - #         45                       - - His His His His His His His His Pro Pro Pr - #o Pro Ala Pro Gln Pro            50             - #     55             - #     60                           - - Pro Pro Pro Pro Pro Gln Gln Gln Gln Gln Pr - #o Pro Pro Ala Pro Gln        65                 - # 70                 - # 75                 - # 80        - - Pro Pro Gln Ala Arg Gly Ala Pro Ala Ala As - #p Asp Asp Lys Gly Pro                        85 - #                 90 - #                 95               - - Gln Pro Leu Leu Leu Pro Pro Ser Ala Ala Le - #u Asp Gly Ala Lys Ala                   100      - #           105      - #           110                   - - Asp Ala Leu Gly Ala Lys Gly Glu Pro Gly Gl - #y Gly Pro Ala Glu Leu               115          - #       120          - #       125                       - - Ala Pro Val Gly Pro Asp Glu Lys Glu Lys Gl - #y Ala Gly Ala Gly Gly           130              - #   135              - #   140                           - - Glu Glu Lys Lys Gly Ala Gly Glu Gly Gly Ly - #s Asp Gly Glu Gly Gly       145                 1 - #50                 1 - #55                 1 -       #60                                                                               - - Lys Glu Gly Asp Lys Asn Asn Gly Lys Tyr Gl - #u Lys Pro Pro Phe         Thr                                                                                              165  - #               170  - #               175              - - Tyr Asn Ala Leu Ile Met Met Ala Ile Arg Gl - #n Ser Pro Glu Lys Arg                   180      - #           185      - #           190                   - - Leu Thr Leu Asn Gly Ile Tyr Glu Phe Ile Me - #t Lys Asn Phe Pro Tyr               195          - #       200          - #       205                       - - Tyr Arg Glu Asn Lys Gln Gly Trp Gln Asn Se - #r Ile Arg His Asn Leu           210              - #   215              - #   220                           - - Ser Leu Asn Lys Cys Phe Val Lys Val Pro Ar - #g His Tyr Asp Asp Pro       225                 2 - #30                 2 - #35                 2 -       #40                                                                               - - Gly Lys Gly Asn Tyr Trp Met Leu Asp Pro Se - #r Ser Asp Asp Val         Phe                                                                                              245  - #               250  - #               255              - - Ile Gly Gly Thr Thr Gly Lys Leu Arg Arg Ar - #g Ser Thr Thr Ser Arg                   260      - #           265      - #           270                   - - Ala Lys Leu Ala Phe Lys Arg Arg Ala Arg Le - #u Thr Ser Thr Gly Leu               275          - #       280          - #       285                       - - Thr Phe Met Asp Arg Ala Gly Ser Leu Tyr Tr - #p Pro Met Ser Pro Phe           290              - #   295              - #   300                           - - Leu Ser Leu His His Pro Arg Ala Ser Ser Th - #r Leu Ser Tyr Asn Gly       305                 3 - #10                 3 - #15                 3 -       #20                                                                               - - Thr Thr Ser Ala Tyr Pro Ser His Pro Met Pr - #o Tyr Ser Ser Val         Leu                                                                                              325  - #               330  - #               335              - - Thr Gln Asn Ser Leu Gly Asn Asn His Ser Ph - #e Ser Thr Ala Asn Gly                   340      - #           345      - #           350                   - - Leu Ser Val Asp Arg Leu Val Asn Gly Glu Il - #e Pro Tyr Ala Thr His               355          - #       360          - #       365                       - - His Leu Thr Ala Ala Ala Leu Ala Ala Ser Va - #l Pro Cys Gly Leu Ser           370              - #   375              - #   380                           - - Val Pro Cys Ser Gly Thr Tyr Ser Leu Asn Pr - #o Cys Ser Val Asn Leu       385                 3 - #90                 3 - #95                 4 -       #00                                                                               - - Leu Ala Gly Gln Thr Ser Tyr Phe Phe Pro Hi - #s Val Pro His Pro         Ser                                                                                              405  - #               410  - #               415              - - Met Thr Ser Gln Thr Ser Thr Ser Met Ser Al - #a Arg Ala Ala Ser Ser                   420      - #           425      - #           430                   - - Ser Thr Ser Pro Gln Ala Pro Ser Thr Leu Pr - #o Cys Glu Ser Leu Arg               435          - #       440          - #       445                       - - Pro Ser Leu Pro Ser Phe Thr Thr Gly Leu Se - #r Gly Gly Leu Ser Asp           450              - #   455              - #   460                           - - Tyr Phe Thr His Gln Asn Gln Gly Ser Ser Se - #r Asn Pro Leu Ile His       465                 4 - #70                 4 - #75                 4 -       #80                                                                               - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                      - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 1860 base - #pairs                                                 (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: cDNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                - - GGCCTTGCCG AGCTCCGTTT CTAGATTCTC ACTCCTCCCG CGCCCTTCTG GG -             #CGCCCGAC     60                                                                  - - ACCGACTGGC CCGCCAGGGT CCAGCCACCC CCTCCTGGAC TAACCGGGCT AA -             #AGGGACCA    120                                                                  - - CAGTGGAAAA GGTCAAGTCT AGAGCGCCCG CCACCCGGTG CCCGCCGAGC CG -             #GGGAGCGC    180                                                                  - - AAACACCTCG CACAGCCCTG CTCGCCAAGT AGCGGAGCGG AGGGGCCCCA GC -             #CACCTCCT    240                                                                  - - GCAGCCGCGC GTCGCAGAGT GGCGTCCTCG CTCCGGGTCC GCCCCTCCGG GA -             #TCGGCCTG    300                                                                  - - GGGAGGCCAG GGAGCCGGGA GCCCGGTGCC CCTATGTGCC GCCGCGCCAC CG -             #CGCCGCCC    360                                                                  - - CAGCTATGAC CCTGAGCACG GAGATGTCCG ATGCCTCCGG CCTCGCGGAG GA -             #GACAGACA    420                                                                  - - TCGACGTGGT GGGGGAGGGC GAGGACGACG AGGAGGAGGA GGACGATGAC GA -             #CGAGGGCG    480                                                                  - - GCGGCGGCCG CGGCGGCGGC GGGTCCCGGT TGCCGAGCTC GGCCCAGCGG CG -             #GAGGCGCT    540                                                                  - - CTTACGCCGG GGAGGTCGAT CTCGAGGACC TGGAGGAGGA GGACGACGAT GA -             #CCTGCTGC    600                                                                  - - TGGCCCCCCG GCCCGCCGCG TCCCCCGCGC CTCCGGGTCC TGCGCCCGCC CC -             #GGGGACGG    660                                                                  - - GGTCGGGCGG CTGCAGCGGC GCCGGAGCGG GAGGCGGCGC GGGAGGTGGT AC -             #GGGCGCGG    720                                                                  - - GCACGGGCGG GGGCGCTAAG AATCCGCTGG TGAAGCCGCC CTACTCGTAC AT -             #CGCGCTCA    780                                                                  - - TCACCATGGC CATCCTGCAG AGCCCCAAGA AGCGCCTGAC GCTCAGCGAG AT -             #CTGCGAGT    840                                                                  - - TCATCAGCAG CCGCTTCCCT TACTACCGGG AGAAGTTCCC CGCTTGGCAG AA -             #CAGCATCC    900                                                                  - - GTCACAACCT GTCGCTCAAC GACTGCTTCG TCAAGATCCC GCGCGAACCG GG -             #CAACCCGG    960                                                                  - - GCAAGGGCAA CTACTGGACG CTCGACCCGG AGTCCGCAGA TATGTTCGAC AA -             #CGGCAGCT   1020                                                                  - - TCCTGCGGCG CCGCAAGCGC TTCAAGCGCC AGCCGCTACT CGATCCCGCG CT -             #GGGGACTC   1080                                                                  - - TGCACCAAGG GACAGCGCTG TCCAGTGTGG AGAACTTTAC TGCTAGGATT TC -             #CAATTGTT   1140                                                                  - - AGGAACGTCG TTAGCGCGCG GGAGAGCGAA GGTAGGACTC CCGGCTTCTT TC -             #TCCGGATG   1200                                                                  - - GGGGGGTTGG TTTCGTTCGC CCCTCCCGGT CCTCGGAGAC CCCGCGCCCC CC -             #GTTTTCGC   1260                                                                  - - CGCTTCGGAT TCTTGGACCA GACTGTGTTG GGCGACAGCT GGGGCGCCGC GC -             #AGTTTAGC   1320                                                                  - - TCAGAGGGTC CATCTATTTA TGCAAAATCG CCCTATGCTG CAACCCTGAC TT -             #GGGGTGGG   1380                                                                  - - AAGGAGGGGA GTCGCTCTGT CTTGGCACTA GGAATTTCCT TGACTTTTGA CA -             #AATTGAGA   1440                                                                  - - AAAAACAAAA CAAAACAAGC AAAATCATCA AAACTAAGCC CTTTTTGAGG TG -             #TAGAGATT   1500                                                                  - - CACAGGTCCA GCGTTTTAAA AAATCAGTAA TGTTTAAATG CAGCTTATAG AA -             #AACCAGTA   1560                                                                  - - AAAGTCTCCA AGAAATGCCT CTACTTGTTC ACACTTGTTT GGTAGACTTT TT -             #TCATGGAA   1620                                                                  - - AGAAAAAAAA TTAACATGTT TACACAAGAA ATAAGTCGAA ATTTACCATT TC -             #CTATTTTT   1680                                                                  - - AACCTGTGTT TTGTATCATA ATGGACATGC GGAATTTTTA TTTTGTACTT AC -             #TACGTATT   1740                                                                  - - CTTTGCAAGG AGTATTGTAA ATTTTACTGG CAATTATTAT TGTACTATTC TA -             #ATGTAAGA   1800                                                                  - - TTTTTACACT TTTTTTCAGA AATAAAATGC TTAATTTTCA AAGAAAAAAA AA -             #AAAAAAAA   1860                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                      - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 1155 base - #pairs                                                 (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: cDNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                - - TCTAGGCTCC ACGGTCGCGC GTGGCGTCTG TGCCGCCAGC TCAGGGCTGC CA -             #CCCGCCAA     60                                                                  - - GCCGAGAGTG CGCGGCCAGC GGGGCCGCCT GCCGTGCACC CTTCAGGATG CC -             #GATCCGCC    120                                                                  - - CGGTCGCTGA ACCCGAGCGC CGGCGTCTTC CGCGCGTGGA CCGCGAGGCT GC -             #CCCGAGTC    180                                                                  - - GGGGCTGCCT GCATCGCTCC GTCCCTTCCT GCTCTCCTGC TCCGGGCCTC GC -             #TCGCCGCG    240                                                                  - - GGCCGCAGTC GGTGCGCGCA GCCGGCGACC GGGCGTCTGG GACACAGCAT GC -             #AGGCGCGT    300                                                                  - - TACTCGGTAT CGGACCCCAA CACCCTGGGA GTGGTACCCT ATTTGAGTGA GC -             #AAAACTAC    360                                                                  - - TACCGGGCGG CCGGCAGCTA CGGCGGCATG GCCAGCCCCA TGGGCGTCTA CT -             #CCGGCCAC    420                                                                  - - CCGGAGCAGT ACGGCGCCGG CATGGGCCGC TCCTACGCGC CCTACCACCA TC -             #AGCCCTTT    480                                                                  - - TCTCCCAAGG ACCTGGTGAA GCCGCCCTAC AGCTATATAG CGCTCATCAC CA -             #TGGCGATC    540                                                                  - - CAGAACGCGC CAGAGAAGAA GATCACTCTG AACGGCATCT ACCAGTTCAT CA -             #TGGACCGT    600                                                                  - - TTCCCCTTCT ACCGCGAGAA CAAGCAGGGC TGGCAGAACA GCATCCGCCA CA -             #ACCTGTCA    660                                                                  - - CTCAATGAGT GCTTCGTGAA AGTGCCGCGC GACGACAAGA AGCCGGGCAA GG -             #GCAGCTAC    720                                                                  - - TGGACGCTCG ACCCGGACTC CTACAACATG TTCGAGAATG GCAGCTTCCT GC -             #GGCGGCGG    780                                                                  - - CGGCGCTTCA AGAAGAAGGA TGTGCCCAAG GACAAGGAGG AGCGGGCCCA CC -             #TCAAGGAG    840                                                                  - - CCGCCCTCGA CCACGGCCAA GGGCGCTCCG ACAGGGACCC CGGTAGCTGA CG -             #GGCCCAAG    900                                                                  - - GAGGCCGAGA AGAAAGTCGT GGTTAAGAGC GAGGCGGCGT CCCCCGCACT GC -             #CGGTCATC    960                                                                  - - ACCAAGGTGG AGACGCTGAG CCCCGAGGGA GCGCTGCAGG CCAGTCCGCG CA -             #GCGCATCC   1020                                                                  - - TCCACGCCCG CAGTGTCCCC AGACGGCTCG CTGCCGGAGC ACCACGCCGC GG -             #CGCCTAAC   1080                                                                  - - GGGCTGCCCG GCTTCAGCGT GGAGACCATC ATGACGCTGC GCACGTCGCC TC -             #CGGGGGCG   1140                                                                  - - ATCTGAGCCC AGCGG              - #                  - #                       - #  1155                                                                 __________________________________________________________________________ 

What is claimed is:
 1. An isolated, animal-derived nucleic encoding Brain Factor-2.
 2. The nucleic acid of claim 1, which is an RNA molecule.
 3. The nucleic acid of claim 1, which is a DNA molecule.
 4. The DNA molecule of claim 3, having Seq. ID No.
 3. 5. A plasmid comprising the DNA of claim
 3. 6. Plasmid pBSKS-BF-2 (ATCC Accession No. 75460).
 7. An isolated, animal-derived nucleic acid encoding Brain Factor-3.
 8. The nucleic acid of claim 7, which is an RNA molecule.
 9. The nucleic acid of claim 7, which is a DNA molecule.
 10. The DNA molecule of claim 9, having Seq. ID No.
 4. 11. A plasmid comprising the DNA of claim
 9. 12. Plasmid BF-3 (ATCC Accession No. 75461). 